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1.
Cells ; 13(12)2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38920678

ABSTRACT

Successful heart development depends on the careful orchestration of a network of transcription factors and signaling pathways. In recent years, in vitro cardiac differentiation using human pluripotent stem cells (hPSCs) has been used to uncover the intricate gene-network regulation involved in the proper formation and function of the human heart. Here, we searched for uncharacterized cardiac-development genes by combining a temporal evaluation of human cardiac specification in vitro with an analysis of gene expression in fetal and adult heart tissue. We discovered that CARDEL (CARdiac DEvelopment Long non-coding RNA; LINC00890; SERTM2) expression coincides with the commitment to the cardiac lineage. CARDEL knockout hPSCs differentiated poorly into cardiac cells, and hPSC-derived cardiomyocytes showed faster beating rates after controlled overexpression of CARDEL during differentiation. Altogether, we provide physiological and molecular evidence that CARDEL expression contributes to sculpting the cardiac program during cell-fate commitment.


Subject(s)
Cell Differentiation , Heart , Homeostasis , Myocytes, Cardiac , RNA, Long Noncoding , Humans , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Cell Differentiation/genetics , Heart/embryology , Heart/physiology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Gene Expression Regulation, Developmental , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Cell Lineage/genetics , Organogenesis/genetics
2.
Front Cell Dev Biol ; 12: 1362671, 2024.
Article in English | MEDLINE | ID: mdl-38425500

ABSTRACT

Producing an adequate number of muscle stem cells (MuSCs) with robust regenerative potential is essential for the successful cell therapy of muscle-wasting disorders. We have recently developed a method to produce skeletal myogenic cells with exceptional engraftability and expandability through an in vivo pluripotent stem cell (PSC) differentiation approach. We have subsequently mapped engraftment and gene expression and found that leukemia inhibitory factor receptor (Lifr) expression is positively correlated with engraftability. We therefore investigated the effect of LIF, the endogenous ligand of LIFR, on cultured MuSCs and examined their engraftment potential. We found that LIF-treated MuSCs exhibited elevated expression of PAX7, formed larger colonies from single cells, and favored the retention of PAX7+ "reserve cells" upon myogenic differentiation. This suggested that LIF promoted the maintenance of cultured MuSCs at a stem cell stage. Moreover, LIF enhanced the engraftment capability of MuSCs that had been expanded in vitro for 12 days by 5-fold and increased the number of MuSCs that repopulated the stem cell pool post-transplantation. These results thereby demonstrated the effectiveness of our in vivo PSC differentiation platform to identify positive regulators of the engraftability of cultured MuSCs.

3.
Sci Rep ; 14(1): 3915, 2024 02 16.
Article in English | MEDLINE | ID: mdl-38365813

ABSTRACT

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure-function. This includes studies centered on disease etiology, drug development, and for potential clinical applications in heart regeneration/repair. Ultimately, for these applications to achieve success, a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state, and this represents a major hurdle for the field. To address this roadblock, we have developed a hiPSC-CMs (ß-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere, the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile, the molecular motor of the heart, which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of α-MyHC in ß-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis, we used gene editing with an inducible α-MyHC expression cassette into isogeneic hiPSC-CMs, and separately by gene transfer, and then investigated the direct effects of increased α-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with α-MyHC. Positive inotropy and relaxation was evident in comparison to ß-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening, as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration, repair and re-muscularization applications.


Subject(s)
Induced Pluripotent Stem Cells , Humans , Ventricular Myosins/genetics , Ventricular Myosins/metabolism , Ventricular Myosins/pharmacology , Gene Editing , Myocardium , Myocytes, Cardiac/metabolism , Cell Differentiation , Myosins/metabolism , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism
4.
Methods Mol Biol ; 2640: 175-189, 2023.
Article in English | MEDLINE | ID: mdl-36995595

ABSTRACT

Generating engraftable skeletal muscle progenitor cells is a promising cell therapy approach to treating degenerating muscle diseases. Pluripotent stem cell (PSC) is an ideal cell source for cell therapy because of its unlimited proliferative capability and potential to differentiate into multiple lineages. Approaches such as ectopic overexpression of myogenic transcription factors and growth factors-directed monolayer differentiation, while able to differentiate PSCs into the skeletal myogenic lineage in vitro, are limited in producing muscle cells that reliably engraft upon transplantation. Here we present a novel method to differentiate mouse PSCs into skeletal myogenic progenitors without genetic modification or monolayer culture. We make use of forming a teratoma, in which skeletal myogenic progenitors can be routinely obtained. We first inject mouse PSCs into the limb muscle of an immuno-compromised mouse. Within 3-4 weeks, α7-integrin+ VCAM-1+ skeletal myogenic progenitors are purified by fluorescent-activated cell sorting. We further transplant these teratoma-derived skeletal myogenic progenitors into dystrophin-deficient mice to assess engraftment efficiency. This teratoma formation strategy is capable of generating skeletal myogenic progenitors with high regenerative potency from PSCs without genetic modifications or growth factors supplementation.


Subject(s)
Pluripotent Stem Cells , Satellite Cells, Skeletal Muscle , Teratoma , Mice , Animals , Satellite Cells, Skeletal Muscle/metabolism , Muscle Fibers, Skeletal/metabolism , Cell Differentiation , Intercellular Signaling Peptides and Proteins , Teratoma/etiology , Muscle, Skeletal/metabolism , Muscle Development
5.
Cells ; 12(3)2023 01 18.
Article in English | MEDLINE | ID: mdl-36766703

ABSTRACT

Differentiation of pluripotent stem cells (PSCs) is a promising approach to obtaining large quantities of skeletal myogenic progenitors for disease modeling and cell-based therapy. However, generating skeletal myogenic cells with high regenerative potential is still challenging. We recently reported that skeletal myogenic progenitors generated from mouse PSC-derived teratomas possess robust regenerative potency. We have also found that teratomas derived from human PSCs contain a skeletal myogenic population. Here, we showed that these human PSC-derived skeletal myogenic progenitors had exceptional engraftability. A combination of cell surface markers, CD82, ERBB3, and NGFR enabled efficient purification of skeletal myogenic progenitors. These cells expressed PAX7 and were able to differentiate into MHC+ multinucleated myotubes. We further discovered that these cells are expandable in vitro. Upon transplantation, the expanded cells formed new dystrophin+ fibers that reconstituted almost ¾ of the total muscle volume, and repopulated the muscle stem cell pool. Our study, therefore, demonstrates the possibility of producing large quantities of engraftable skeletal myogenic cells from human PSCs.


Subject(s)
Pluripotent Stem Cells , Satellite Cells, Skeletal Muscle , Teratoma , Humans , Animals , Mice , Pluripotent Stem Cells/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Muscle Fibers, Skeletal , Cell Differentiation , Nerve Tissue Proteins/metabolism , Receptors, Nerve Growth Factor/metabolism , Kangai-1 Protein/metabolism , Receptor, ErbB-3/metabolism
6.
Dev Cell ; 57(18): 2181-2203.e9, 2022 09 26.
Article in English | MEDLINE | ID: mdl-36108627

ABSTRACT

Many developmental signaling pathways have been implicated in lineage-specific differentiation; however, mechanisms that explicitly control differentiation timing remain poorly defined in mammals. We report that murine Hedgehog signaling is a heterochronic pathway that determines the timing of progenitor differentiation. Hedgehog activity was necessary to prevent premature differentiation of second heart field (SHF) cardiac progenitors in mouse embryos, and the Hedgehog transcription factor GLI1 was sufficient to delay differentiation of cardiac progenitors in vitro. GLI1 directly activated a de novo progenitor-specific network in vitro, akin to that of SHF progenitors in vivo, which prevented the onset of the cardiac differentiation program. A Hedgehog signaling-dependent active-to-repressive GLI transition functioned as a differentiation timer, restricting the progenitor network to the SHF. GLI1 expression was associated with progenitor status across germ layers, and it delayed the differentiation of neural progenitors in vitro, suggesting a broad role for Hedgehog signaling as a heterochronic pathway.


Subject(s)
Gene Regulatory Networks , Hedgehog Proteins , Animals , Cell Differentiation/genetics , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Mice , Signal Transduction/physiology , Zinc Finger Protein GLI1/genetics
7.
Cells ; 11(9)2022 05 09.
Article in English | MEDLINE | ID: mdl-35563894

ABSTRACT

Skeletal muscle stem cells are essential to muscle homeostasis and regeneration after injury, and have emerged as a promising cell source for treating skeletal disorders. An attractive approach to obtain these cells utilizes differentiation of pluripotent stem cells (PSCs). We recently reported that teratomas derived from mouse PSCs are a rich source of skeletal muscle stem cells. Here, we showed that teratoma formation is also capable of producing skeletal myogenic progenitors from human PSCs. Using single-cell transcriptomics, we discovered several distinct skeletal myogenic subpopulations that represent progressive developmental stages of the skeletal myogenic lineage and recapitulate human embryonic skeletal myogenesis. We further discovered that ERBB3 and CD82 are effective surface markers for prospective isolation of the skeletal myogenic lineage in human PSC-derived teratomas. Therefore, teratoma formation provides an accessible model for obtaining human skeletal myogenic progenitors from PSCs.


Subject(s)
Pluripotent Stem Cells , Teratoma , Animals , Humans , Mice , Muscle Development/physiology , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal , Pluripotent Stem Cells/metabolism
8.
Stem Cell Reports ; 16(12): 2900-2912, 2021 12 14.
Article in English | MEDLINE | ID: mdl-34798067

ABSTRACT

One major challenge in realizing cell-based therapy for treating muscle-wasting disorders is the difficulty in obtaining therapeutically meaningful amounts of engraftable cells. We have previously described a method to generate skeletal myogenic progenitors with exceptional engraftability from pluripotent stem cells via teratoma formation. Here, we show that these cells are functionally expandable in vitro while retaining their in vivo regenerative potential. Within 37 days in culture, teratoma-derived skeletal myogenic progenitors were expandable to a billion-fold. Similar to their freshly sorted counterparts, the expanded cells expressed PAX7 and were capable of forming multinucleated myotubes in vitro. Importantly, these cells remained highly regenerative in vivo. Upon transplantation, the expanded cells formed new DYSTROPHIN+ fibers that reconstituted up to 40% of tibialis anterior muscle volume and repopulated the muscle stem cell pool. Our study thereby demonstrates the possibility of producing large quantities of engraftable skeletal myogenic cells for transplantation.


Subject(s)
Induced Pluripotent Stem Cells/pathology , Muscle Development , Muscle, Skeletal/pathology , Stem Cell Transplantation , Teratoma/pathology , Animals , Cell Compartmentation , Cell Differentiation , Cell Proliferation , Mice , Muscle Fibers, Skeletal , RNA-Seq , Vascular Cell Adhesion Molecule-1/metabolism
9.
Dev Growth Differ ; 62(7-8): 487-494, 2020 Oct.
Article in English | MEDLINE | ID: mdl-33048365

ABSTRACT

Efficient derivation of cardiomyocytes from mouse pluripotent stem cells has proven challenging, and existing approaches rely on expensive supplementation or extensive manipulation. Mesp1 is a transcription factor that regulates cardiovascular specification during embryo development, and its overexpression has been shown to promote cardiogenesis. Here, we utilize a doxycycline-inducible Mesp1-expressing mouse embryonic stem cell system to develop an efficient differentiation protocol to generate functional cardiomyocytes. Our cardiac differentiation method involves transient Mesp1 induction following by subsequent dual inhibition of TGFß and Wnt signaling pathways using small molecules. We discovered that whereas TGFß inhibition promoted Mesp1-induced cardiac differentiation, Wnt inhibition was ineffective. Nevertheless, a combined inhibition of both pathways was superior to either inhibition alone in generating cardiomyocytes. These observations suggested a potential interaction between TGFß and Wnt signaling pathways in the context of Mesp1-induced cardiac differentiation. Using a step-by-step approach, we have further optimized the windows of Mesp1 induction, TGFß inhibition and Wnt inhibition to yield a maximal cardiomyocyte output - Mesp1 was induced first, followed by dual inhibition of TGFß and Wnt signaling. Our protocol is capable of producing approximately 50% of cardiomyocytes in 12 days, which is comparable to existing methods, and have the advantages of being technically simple and inexpensive. Moreover, cardiomyocytes thus derived are functional, displaying intrinsic contractile capacity and contraction in response to electric stimulus. Derivation of mouse cardiomyocytes without the use of growth factors or other costly supplementation provides an accessible cell source for future applications.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation , Mouse Embryonic Stem Cells/metabolism , Myocytes, Cardiac , Transforming Growth Factor beta/metabolism , Wnt Signaling Pathway , Animals , Mice
10.
Biochem Biophys Res Commun ; 520(2): 284-290, 2019 12 03.
Article in English | MEDLINE | ID: mdl-31590918

ABSTRACT

The Mesp1 lineage contributes to cardiac, hematopoietic and skeletal myogenic development. Interestingly, muscle stem cells residing in craniofacial skeletal muscles primarily arise from Mesp1+ progenitors, but those in trunk and limb skeletal muscles do not. To gain insights into the difference between the head and trunk/limb muscle developmental processes, we studied Mesp1+ skeletal myogenic derivatives via single-cell RNA-seq and other strategies. Using a doxycycline-inducible Mesp1-expressing mouse embryonic stem cell line, we found that the development of Mesp1-induced skeletal myogenic progenitors can be characterized by dynamic expression of PDGFRα and VCAM1. Single-cell RNA-seq analysis further revealed the heterogeneous nature of these Mesp1+ derivatives, spanning pluripotent and mesodermal to mesenchymal and skeletal myogenic. We subsequently reconstructed the single-cell trajectories of these subpopulations. Our data thereby provide a cell fate projection of Mesp1-induced skeletal myogenesis.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Muscle, Skeletal/metabolism , RNA-Seq , Single-Cell Analysis , Animals , Anti-Bacterial Agents/pharmacology , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/drug effects , Cells, Cultured , Doxycycline/pharmacology , Mice , Muscle Development/drug effects , Muscle, Skeletal/drug effects
11.
Reumatismo ; 71(2): 108-112, 2019 Jul 09.
Article in English | MEDLINE | ID: mdl-31309785

ABSTRACT

Systemic lupus erythematosus (SLE) is a chronic inflammatory multisystem autoimmune disease. Ascites when associated with pleural effusion and raised CA-125 levels in SLE patient, is known as pseudo-pseudo Meigs' syndrome (PPMS). This is the case of a 22-year-old lady who presented with complaints of abdominal distension for one month and had a history of spontaneous abortion in the past. Abdominal imaging did not reveal any tumor and after extensive workup a diagnosis of PPMS was made. She was successfully treated with steroids, hydroxychloroquine and cyclophosphamide.


Subject(s)
Lupus Erythematosus, Systemic/complications , Meigs Syndrome/etiology , Female , Humans , Lupus Erythematosus, Systemic/diagnosis , Young Adult
12.
J Physiol ; 596(19): 4665-4680, 2018 10.
Article in English | MEDLINE | ID: mdl-30035314

ABSTRACT

KEY POINTS: The female hormone oestrogen may protect muscle from injury by reducing inflammation but this is debatable. In this study, the inflammatory response of injured muscle from oestrogen-replete mice was comprehensively compared to that from oestrogen-deficient mice. We show that oestrogen markedly promotes movement of neutrophils, an inflammatory white blood cell type, into muscle over the first few days after injury but has only a minor effect on the movement of macrophages, another inflammatory cell type. Despite the enhancement of inflammation by oestrogen in injured muscle, we found strength in oestrogen-replete mice to recover faster and to a greater extent than it does in oestrogen-deficient mice. Our study and others indicate that lower doses of oestrogen, such as that used in our study, may affect muscle inflammation and injury differently from higher doses. ABSTRACT: Oestrogen has been shown to protect against skeletal muscle injury and a reduced inflammatory response has been suggested as a possible protective mechanism. There are, however, dissenting reports. Our objective was to conduct an unbiased, comprehensive study of the effect of oestradiol on the inflammatory response following muscle injury. Female C57BL6/J mice were ovariectomized and supplemented with and without oestradiol. Tibialis anterior muscles were freeze injured and studied primarily at 1-4 days post-injury. Oestradiol supplementation increased injured muscle gene expression of neutrophil chemoattractants (Cxcl1 and Cxcl5) and to a lesser extent that of monocyte/macrophage chemoattractants (Ccl2 and Spp1). Oestradiol markedly increased gene expression of the neutrophil cell surface marker (Ly6g) but had less consistent effects on the monocyte/macrophage cell surface markers (Cd68, Cd163 and Cd206). These results were confirmed at the protein level by immunoblot with oestradiol increasing LY6G/C content and having no significant effect on CD163 content. These findings were confirmed with fluorescence-activated cell sorting counts of neutrophils and macrophages in injured muscles; oestradiol increased the proportion of CD45+ cells that were neutrophils (LY6G+ ) but not the proportion that were macrophages (CD68+ or CD206+ ). Physiological impact of the oestradiol-enhanced neutrophil response was assessed by strength measurements. There was no significant difference in strength between oestradiol-supplemented and -unsupplemented mice until 2 weeks post-injury; strength was 13-24% greater in supplemented mice at 2-6 weeks post-injury. In conclusion, a moderate level of oestradiol supplementation enhances neutrophil infiltration in injured muscle and this is associated with a beneficial effect on strength recovery.


Subject(s)
Estradiol/metabolism , Inflammation/prevention & control , Muscle Strength , Muscle, Skeletal/physiology , Muscular Diseases/prevention & control , Neutrophils/physiology , Recovery of Function , Animals , Biomarkers/analysis , Chemokine CCL11/genetics , Chemokine CCL11/metabolism , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Chemokine CXCL5/genetics , Chemokine CXCL5/metabolism , Estrogens , Female , Gene Expression Profiling , Inflammation/immunology , Inflammation/metabolism , Macrophages/cytology , Macrophages/immunology , Macrophages/physiology , Mice , Mice, Inbred C57BL , Muscle, Skeletal/immunology , Muscle, Skeletal/injuries , Muscle, Skeletal/metabolism , Muscular Diseases/immunology , Muscular Diseases/metabolism , Neutrophils/cytology , Neutrophils/immunology , Osteopontin/genetics , Osteopontin/metabolism
13.
Nat Commun ; 9(1): 856, 2018 02 22.
Article in English | MEDLINE | ID: mdl-29472544

ABSTRACT

In the originally published version of this Article, an incorrect grant number, RO1 NS083549, was acknowledged. The correct grant number is RO1 AR055685. This error has now been corrected in both the PDF and HTML versions of the Article.

14.
Nat Commun ; 8(1): 550, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28916757

ABSTRACT

Facioscapulohumeral muscular dystrophy is a slowly progressive but devastating myopathy caused by loss of repression of the transcription factor DUX4; however, DUX4 expression is very low, and protein has not been detected directly in patient biopsies. Efforts to model DUX4 myopathy in mice have foundered either in being too severe, or in lacking muscle phenotypes. Here we show that the endogenous facioscapulohumeral muscular dystrophy-specific DUX4 polyadenylation signal is surprisingly inefficient, and use this finding to develop an facioscapulohumeral muscular dystrophy mouse model with muscle-specific doxycycline-regulated DUX4 expression. Very low expression levels, resulting in infrequent DUX4 + myonuclei, evoke a slow progressive degenerative myopathy. The degenerative process involves inflammation and a remarkable expansion in the fibroadipogenic progenitor compartment, leading to fibrosis. These animals also show high frequency hearing deficits and impaired skeletal muscle regeneration after injury. This mouse model will facilitate in vivo testing of therapeutics, and suggests the involvement of fibroadipogenic progenitors in facioscapulohumeral muscular dystrophy.Facioscapulohumeral muscular dystrophy is a severe myopathy that is caused by abnormal activation of DUX4, and for which a suitable mouse model does not exist. Here, the authors generate a novel mouse model with titratable expression of DUX4, and show that it recapitulates several features of the human pathology.


Subject(s)
Homeodomain Proteins/metabolism , Muscle, Skeletal/metabolism , Muscular Dystrophy, Facioscapulohumeral/metabolism , Animals , Disease Models, Animal , Female , Homeodomain Proteins/genetics , Humans , Male , Mice , Muscle, Skeletal/pathology , Muscular Dystrophy, Facioscapulohumeral/genetics , Muscular Dystrophy, Facioscapulohumeral/pathology , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism
15.
Stem Cell Reports ; 9(1): 12-22, 2017 07 11.
Article in English | MEDLINE | ID: mdl-28528701

ABSTRACT

Recent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes, they have not demonstrated scalability, safety, and in vivo engraftment, which are key aspects for their future clinical application. Here we recapitulate one prominent protocol, and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation, the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle, we incorporated the inducible PAX7 lentiviral system into this protocol, which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7+ myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases.


Subject(s)
Cell Culture Techniques/methods , Muscle Cells/cytology , Muscle Cells/transplantation , Muscle Development , PAX7 Transcription Factor/genetics , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation , Cell Line , Cell Proliferation , Cells, Cultured , Humans , Lentivirus/genetics , Male , Mice , Muscle Cells/metabolism , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/physiology , Pluripotent Stem Cells/metabolism , Regeneration , Transgenes
16.
Stem Cell Reports ; 3(4): 594-605, 2014 Oct 14.
Article in English | MEDLINE | ID: mdl-25358788

ABSTRACT

There is no consensus in the stem cell field as to what constitutes the mature cardiac myocyte. Thus, helping formalize a molecular signature for cardiac myocyte maturation would advance the field. In the mammalian heart, inactivation of the "fetal" TNNI gene, TNNI1 (ssTnI), together in temporal concert with its stoichiometric replacement by the adult TNNI gene product, TNNI3 (cTnI), represents a quantifiable ratiometric maturation signature. We examined the TNNI isoform transition in human induced pluripotent stem cell (iPSC) cardiac myocytes (hiPSC-CMs) and found the fetal TNNI signature, even during long-term culture. Rodent stem cell-derived and primary myocytes, however, transitioned to the adult TnI profile. Acute genetic engineering of hiPSC-CMs enabled a rapid conversion toward the mature TnI profile. While there is no single marker to denote the mature cardiac myocyte, we propose that tracking the cTnI:ssTnI protein isoform ratio provides a valuable maturation signature to quantify myocyte maturation status across laboratories.


Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Troponin I/metabolism , Animals , Cells, Cultured , Embryonic Stem Cells/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Mice , Myocytes, Cardiac/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Rats , Rats, Sprague-Dawley , Troponin I/genetics
17.
Methods Mol Biol ; 660: 201-11, 2010.
Article in English | MEDLINE | ID: mdl-20680821

ABSTRACT

Mounting evidence suggests the regenerative potential of the mammalian heart. Nevertheless, the contribution of endogenous stem or precursor cells to adult cardiac regeneration upon myocardial injuries remains unclear. We hereby describe a genetic fate-mapping approach to study adult cardiomyocyte replenishment after myocardial injury. Using double transgenic MerCreMer-ZEG mice, the fate of adult cardiomyocytes can be tracked by the expression of green fluorescence protein (GFP) specifically induced in cardiomyocytes. Upon experimental myocardial infarction, a reduction in GFP expression in the myocardium is observed, indicating the refreshment of cardiomyocytes by endogenous stem or precursor cells.


Subject(s)
Myocardial Ischemia/pathology , Myocytes, Cardiac/cytology , Animals , Genotype , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunohistochemistry , Mice , Mice, Transgenic , Microscopy, Fluorescence , Myocardial Ischemia/metabolism , Myocytes, Cardiac/physiology , Polymerase Chain Reaction
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